1. Field of the Invention
The present invention relates to antibodies, including specified portions or variants, specific for at least one human monocyte chemoattractant protein-1 (MCP-1) protein or fragment thereof, as well as anti-idiotype antibodies, and nucleic acids encoding such anti-MCP-1 antibodies, complementary nucleic acids, vectors, host cells, and methods of making and using thereof, including therapeutic formulations, administration and devices.
2. Related Art
The Human Monocyte Chemoattractant Protein-1 (MCP-1) (also called CCL-2), a 8.6 kDa protein containing 76 amino acid residues, is a member of the chemokine-beta (or C—C) family of cytokines. Chemokines are low molecular weight (8-10 kDa), inducible, secreted, pro-inflammatory, chemotactic cytokines that have been shown to play a central role in the peri-vascular transmigration and accumulation of specific subsets of leukocytes at sites of tissue damage. Two major families have been defined depending on the positioning of four conserved cysteines. The CXC or α-chemokines predominantly attract neutrophils, whereas the CC or β-chemokines predominantly attract monocytes and other leukocytes but not neutrophils (Leonard and Yoshimura et al., 1990). Members of the Monocyte Chemotactic Protein-1 (MCP-1) family form a major component of the C—C family of chemokines and are considered the principal chemokines involved in the recruitment of monocytes, macrophages, and activated lymphocytes. Looking at the homology of MCP-1 from different species, the human and the monkey MCP-1 differ in 2 amino acids only, revealing a sequence identity of 97%, while murine MCP-1, a 13.8 kDa protein containing 125 amino acid residues, differs from human MCP-1 in molecular size and extent of glycosylation.
Chemokine receptors belong to the large family of G protein-coupled, seven transmembrane (7 TM) domain receptors (GPCRs, also called serpentine receptors). Based on the receptor nomenclature established at the 1996 Gordon Research Conference on chemotactic cytokines, the chemokine receptors that bind CXC chemokines are designated CXCRs and the receptors that bind CC chemokines are designated CCRs.
MCP-1 is known to bind and signal through the chemokine receptor CCR2. CCR2 is a seven trans-membrane-spanning G-protein-coupled receptor expressed on many cells including monocytes, T-cells, B-cells, and basophils. Two MCP-1 specific receptors, CCR2A and CCR2B, have been cloned which signal in response to nanomolar (nM) concentrations of MCP-1. CCR2A (CC-CKR2A) and CCR2B (CC-CKR2A) represent two cDNAs that encode two MCP-1-specific receptors with alternatively spliced carboxyl tails. MCP-1 binds to both isoforms with high affinity MCP-1 induces calcium flux in cells expressing CCR2B but not in cells expressing CCR2A. 5-fold less MCP-1 induces chemotaxis in cells expressing CCR2B compared to cells expressing CCR2A.
Other proteins with certain functional and sequence homology to human MCP-1 are known. Especially similar to MCP-1 (GenBank NP—002973) are MCP-2 (GenBank NP—005614) and eotaxin (GenBank P—51671); MCP-2 having 61.8 percent and eotaxin-1 having 63.2 percent sequence identity to MCP-1. The range of activities and spectrum of involvement of these proteins in human homeostatic mechanisms and pathology is not as well understood for the homologs of MCP-1. For example, MCP-2 (renamed CCL8) is related closely to MCP-1 and MCP-3 (renamed CCL7, Genbank NP—006264) and uses both CCR1 as well as CCR2B as its functional receptors. MCP-3 binds to a receptor designated D6. MCP-3 also binds to CCR10 and CCR1. The MCP-3 protein (97 amino acids) sequence shows 74 percent identity with MCP-1 and 58 percent homology with MCP-2. Secreted MCP-3 differs from MCP-1 in being N-glycosylated. MCP-4 (renamed CCL13, Genbank NP—005399) shares 56-61 percent sequence identity with the three known monocyte chemotactic proteins and is 60 percent identical with Eotaxin-1. The functions of MCP-4 appear to be highly similar to those of MCP-3 and Eotaxin. Like MCP-3, MCP-4 is a potent chemoattractant for monocytes and T-lymphocytes. It is inactive on neutrophils. On monocytes, MCP-4 binds to receptors that recognize MCP-1, MCP-3, RANTES (CCL5), and eotaxin (the CCR1 and CCR3 receptors) and shows full cross-desensitization with eotaxin-1. MCP-5 is murine CC-chemokine and related most closely to human MCP-1 (66% amino acid identity). The gene symbol for MCP-5 is SCYA12 (renamed CCL12). Cells transfected with the chemokine receptor CCR2 have been shown to respond to MCP-5. For general information on cytokines and chemokines see www._copewithcytokines.de/cope.cgi and for the current classification system, Zlotnik A., Yoshie O. 2000. Chemokines: a new classification system and their role in immunity. Immunity 12: 121-127.
125I-MCP-1 binds to monocytes and Scatchard plot analysis indicated that monocytes had a minimum of ˜1700 binding sites per cell with a Kd of ˜2 nM (Yoshimura and Leonard, 1990). Later equilibrium binding experiments with human monocytes reveal the presence of approximately 3000 binding sites per cell with a Kd of 0.77 nM (Ernst et al., 1994). 125I-MCP-1 also demonstrated high-affinity binding to dEoL-3 cells expressing CCR2 receptor with a Kd value of 0.4 nM (Sarau et al., 1997) confirming the sub-nanomolar affinity of MCP-1 to its receptor. To identify the regions of MCP-1 that contact its receptor, CCR2, all surface-exposed residues were substituted with alanine. Some residues were also mutated to other amino acids to identify the importance of charge, hydrophobicity, or aromaticity at specific positions. Two clusters of primarily basic residues (R24, K35, K38, K49, and Y13), separated by a 35 A hydrophobic groove, reduced the level of binding by 15-100-fold. Data suggest a model in which a large surface area of MCP-1 contacts the receptor, and the accumulation of a number of weak interactions results in the 35 pM affinity observed for the wild-type (WT) protein (Hemmerich et al., 1999). The range of affinities from 2 nM down to 35 pM in the literature might be due to the assays used and the respective assay limitations.
Other proteins with certain functional and sequence homology to human MCP-1 are known. Especially similar to MCP-1 (GenBank NP—002973) are MCP-2 (GenBank NP—005614) and eotaxin (GenBank P—51671); MCP-2 having 61.8 percent and eotaxin-1 having 63.2 percent sequence identity to MCP-1. The range of activities and spectrum of involvement of these proteins in human homeostatic mechanisms and pathology is not as well understood for the homologs of MCP-1. For example, MCP-2 is related closely to MCP-1 and MCP-3 (Genbank NP—006264) and uses both CCR1 as well as CCR2B as its functional receptors. MCP-3 binds to a receptor designated D6. MCP-3 also binds to CCR10. The MCP-3 protein (97 amino acids) sequence shows 74 percent identity with MCP-1 and 58 percent homology with MCP-2. Secreted MCP-3 differs from MCP-1 in being N-glycosylated. MCP-4 (Genbank NP—005399) shares 56-61 percent sequence identity with the three known monocyte chemotactic proteins and is 60 percent identical with Eotaxin-1. The functions of MCP-4 appear to be highly similar to those of MCP-3 and Eotaxin. Like MCP-3, MCP-4 is a potent chemoattractant for monocytes and T-lymphocytes. It is inactive on neutrophils. On monocytes MCP-4 binds to receptors that recognize MCP-1, MCP-3, and RANTES (CCR2). On eosinophils MCP-4 has similar efficacy and potency as MCP-3, RANTES, and Eotaxin. MCP-4 shares receptors with eotaxin (CCR1 and CCR3) and shows full cross-desensitization with eotaxin-1.
Other antibodies capable of binding MCP-1 have been reported: JP9067399 discloses an antibody obtained from isolated blood cells and JP05276986 discloses a hybridoma secreting an IgM anti-human MCP-1. More recently, antibodies capable of binding a plurality of beta-chemokines including MCP-1 were disclosed (WO03048083) and an MCP-1 binding antibody which also binds eotaxin (US20040047860).
Accordingly, there is a need to provide human antibodies specific for human MCP-1 for use in therapy to diminish or eliminate symptoms of MCP-1-dependent diseases, as well as improvements over known antibodies or fragments thereof.